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Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) and foreign (streptavidin) components, conjugated to
biotinylated virus-like particles (VLPs). Similar titers of
autoantibodies to TNF- were elicited using conjugated polyomavirus
VLPs and papillomavirus VLPs, indicating that acute activation of
dendritic cells by the Ag is not required. Strong autoantibody
responses were also induced by conjugated papillomavirus capsid
pentamers, indicating that a higher order particulate structure is also
not required. However, a reduction of self-Ag density on VLP surfaces
dramatically reduced the efficiency of IgG autoantibody induction. In
contrast, the negative effects of reductions in foreign Ag density were
limited and could be overcome by dosage and adjuvant. These data
suggest that the immune system has evolved to differentially recognize
closely spaced repetitive Ags and that the signals generated upon
interactions with high-density self-Ags can overwhelm the normal
mechanisms for B cell tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been proposed that repetitive determinants at a spacing of
50–100 Å are unique to microbial Ags and, therefore, the mammalian
immune system has evolved to respond strongly to this arrangement of
epitopes (13, 14, 15). Using immunogens in which repetitively
arranged self-antigenic epitopes are arrayed on the surface of virus or
virus-like particles, strong autoantibody responses have been elicited
against either an introduced transgene (16, 17) or
self-Ags (11, 12, 18). For example, immunization with a
model Ag containing self (TNF-) and nonself (streptavidin (SA))
fusion domains, conjugated to biotinylated papillomavirus VLPs, induced
a 1000-fold increase in autoantibody titers, relative to animals
injected with the fusion protein alone (12).
Coadministration of a strong adjuvant, such as CFA, could complement,
but not substitute for, conjugation to VLPs. In addition, immunization
with VLP-conjugated Ag markedly diminished humoral immune distinctions
between the self and nonself domains of the fusion protein.
Although it has been established that conjugated virus particles are able to circumvent the normal mechanisms of B cell tolerance, it is unclear whether the particulate nature of the Ag, the density of the repeated antigenic epitopes, or specific activation of immune cells by virus particles plays the dominant role in breaking B cell tolerance. Papillomavirus VLPs are intrinsically immunogenic; systemic vaccination of VLPs without adjuvant induces high titers of neutralizing Abs and strong CTL responses (19, 20, 21, 22, 23). This immunogenicity may be related to the ability of papillomavirus VLPs to avidly bind and potently activate dendritic cells (DCs) in vitro (24, 25). Thus, it is possible that activated DCs play a role in subsequent activation of autoreactive B cells, because interactions with DCs have been implicated in B cell differentiation and isotype switching (26).
Therefore, we set out to systematically explore the structural
correlates of autoantibody induction. By taking advantage of the
ability to conjugate the SA-TNF- Ag onto diverse virion-derived
structures at different levels of occupancy, we examined the relative
contributions of particle type, size, and Ag density in the induction
of Abs against self and foreign Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VLPs were generated from recombinant baculovirus-infected Sf9 cell cultures, as described previously (27). We generated papillomavirus VLPs composed of the L1 major capsid protein from the strains human papillomavirus (HPV)16(K), the assembly-deficient mutant HPV16(P), and bovine papillomavirus (BPV). Human polyomavirus VLPs were composed of the BK virus (BKV) VP1 major capsid protein (24, 28). VLPs were biotinylated by incubation with NHS-LC-Biotin (Pierce Endogen, Rockford, IL) at a 1:2 (weight:weight) ratio for 60 min at room temperature. Biotinylated VLPs were purified by layering the mixture onto a 24–54% linear sucrose gradient, followed by ultracentrifugation (200,000 x g, 3 h) using an SW-40 rotor (Beckman Instruments, Palo Alto, CA). Fractions were harvested from the bottom and analyzed by SDS-PAGE and Coomassie blue staining for the presence of L1 or VP1. VLP-containing fractions were pooled and quantitated.
Preparation of conjugated VLPs
The SA-TNF- fusion construct
SA-TNF-3–22 was engineered, expressed in
bacteria, and purified from inclusion bodies, as described previously
(12). SA-TNF-3–22 was purified
to homogeneity by applying soluble protein to a 2-iminobiotin-agarose
column (Pierce Endogen) and following the manufacturer’s instructions.
The active concentration of SA-TNF- fusion protein was determined by
ELISA by measuring reactivity to biotinylated BSA (Pierce Endogen)
immobilized (at 200 ng/well) on Immulon II microtiter plates (Dynex
Technologies, Chantilly, VA), as described previously
(12). Conjugated VLPs were prepared by incubating
biotinylated VLPs with saturating amounts (unless stated otherwise) of
SA-TNF-3–22, rSA (Zymed Laboratories, South
San Francisco, CA), or a mixture of
SA-TNF-3–22 and rSA for 1 h at room
temperature with gentle rocking. Conjugated particles were separated
from unconjugated SA-TNF- fusion protein using a MidJet cross flow
filtration system and MidGee cartridges with a 500-kDa pore size (A/G
Technologies, Needham, MA).
Detection of SA-TNF- binding to biotinylated VLPs
The relative ability of SA-TNF-3–22
and rSA to bind biotinylated BPV-L1 VLPs was measured by ELISA.
Biotinylated BPV-L1 VLPs were immobilized (at 150 ng/well) on Immulon
II microtiter plates (Dynex Technologies). After blocking with
PBS-0.5% nonfat dry milk (50 µl) for 1 h at room temperature,
100 ng of a mixture of rSA and SA-TNF-3–22
were reacted in 50 µl for 1.5 h at room temperature with the
immobilized biotinylated VLPs with gentle rocking. After five washes
with PBS, specific binding to biotinylated VLPs by
SA-TNF-3–22 was detected using a rabbit
anti-mouse TNF- polyclonal Ab (50 µl; Pierce Endogen) at a
1/1000 dilution in PBS-0.5% milk for 1 h at room temperature.
After three washes with PBS, HRP-labeled goat-anti-rabbit IgG (50
µl; Boehringer Mannheim, Indianapolis, IN) was added at a 1/3000
dilution in PBS-0.5% milk for 1 h at room temperature. After
three washes with PBS, the substrate ABTS (Boehringer Mannheim) was
added, and OD405 was read. Data is based on the
average OD of triplicate wells. SA-TNF-3–22
binding to biotinylated VLPs in the presence of rSA was also
demonstrated by sucrose gradient centrifugation (using the conditions
described above). Fractions were applied to nitrocellulose membranes by
slot blot and assayed for L1 or TNF- by Western blot using the
TNF- polyclonal Ab mentioned previously, or an anti-L1 mAb
(MAB837; 1/5000 dilution; Chemicon International, Temecula,
CA).
Inoculations
Antiserum was prepared by inoculating 6- to 8-wk-old C57BL/6 mice with conjugated particle preparations. Mice were inoculated i.m. three times at 2-wk intervals. When CFA was used, Ag was diluted 1/1 in CFA for the initial injection, and diluted 1/1 in IFA for subsequent inoculations. Sera were collected at each injection and 2 wk after the final boost. Animal care was in accordance with National Institutes of Health guidelines.
ELISA analysis of Ab induction
Mouse Abs to biologically active recombinant mouse TNF-
(PeproTech, Rocky Hill, NJ) or rSA (Zymed Laboratories) were detected
by ELISA using 200 ng of the target protein per well, as described
(12). Briefly, mouse serum was serially diluted in
PBS-0.5% milk and applied to wells. Reactivity to target Ag was
determined using a 1/2000 dilution of HRP-labeled goat anti-mouse
IgG (BD PharMingen, San Diego, CA). Upon development,
OD405 was read, using a ThermoMax microplate
reader. OD405 values that were greater than twice
background (usually >0.1) were considered positive.
Isolation and stimulation of bone marrow-derived DCs (BMDCs)
BMDCs were generated from bone marrow cells of C57BL/6 mice by negative selection of lineage marker-positive cells using magnetic beads (anti-CD4 (L3T4), anti-CD8a (Ly-2), anti-CD19, and anti-NK cell (DX5) MicroBeads; Miltenyi Biotec, Auburn, CA) and culture in murine rGM-CSF (1000 U/ml) and murine rIL-4 (1000 U/ml; both BD PharMingen) (29). After 6 days at 37°C, cells were harvested and preparations contained 67–88% DCs, as assessed by CD11c expression. The day-6 BMDCs were replated at 1 x 106 cells/ml in 24-well plates and exposed to BPV-L1 VLPs (10 µg/ml), rSA-conjugated BPV-L1 VLPs (10 µg/ml), or rSA (10 µg/ml) for 24 h. LPS (Escherichia coli 026:b6; Sigma-Aldrich, St. Louis, MO) (1 µg/ml) served as positive control for DC maturation.
Flow cytometry
Cells were collected into cold HBSS without Phenol Red (Life Technologies, Grand Island, NY) plus 0.1% BSA (Life Technologies) and 0.1% sodium azide (Sigma-Aldrich). To avoid nonspecific binding of labeled Ab, FcBlock (purified rat anti-mouse CD16/CD32; BD PharMingen) was added at 1 µg for 106 cells. Double staining was performed using mAbs against the following mouse surface Ags (all BD PharMingen): FITC-CD40 (3/23, rat IgG2a), FITC-CD54 (3E2, hamster IgG), FITC-CD86 (GL1, rat IgG2a), PE-CD11c (HL3, hamster IgG), and appropriate control Abs. A total of 105 cells per assay were incubated with the respective Abs at a concentration of 2.5 µg/sample for 30–45 min at 4°C. After two rounds of washings, cellular fluorescence was monitored by flow cytometry using a FACSCalibur instrument and analyzed using CellQuest software (both BD Biosciences, Mountain View, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine whether the ability to induce autoantibodies is a
common characteristic of naked icosahedral VLPs with repetitive
structure or whether papillomavirus VLPs have a unique capacity, mice
were immunized with self-Ag conjugated to human polyomavirus BK (BKV)
VLPs or BPV VLPs. Polyomavirus VLPs are morphologically similar, but
genetically unrelated, to papillomavirus VLPs. Both VLPs are composed
of 72 pentamers of a major capsid protein (L1 for papillomaviruses and
VP1 for polyomaviruses) arranged in T = 7 symmetry, although
polyomavirus VLPs are slightly smaller in diameter (45–50 nm vs 55–60
nm for papillomavirus VLPs) (30, 31, 32). However, unlike
papillomavirus VLPs, polyomavirus VLPs do not induce the maturation of
mouse BMDCs (24). Using the system that we developed
previously to make conjugated particles, BPV-L1 VLPs and BKV-VP1 VLPs
were biotinylated and conjugated with saturating amounts of a SA-(mouse
TNF-) fusion protein that had been demonstrated to induce high-titer
autoantibodies to mouse TNF- when conjugated to BPV-L1 VLPs. Groups
of three C57BL/6 mice were immunized with three doses of 5 µg of
conjugated VLPs, with and without coadministration of CFA. Two
weeks after the final boost, sera were taken from immunized mice, and
IgG Abs specific for the foreign (SA) and self (TNF-) Ag components
of the vaccine were measured by ELISA using native full-length rSA and
rTNF- proteins as the target Ags. Conjugated BKV-VP1 VLPs
elicited similar Ab responses as conjugated BPV-L1 VLPs (Fig. 1
). Both conjugated VLPs, when given
without exogenous adjuvant, induced high-titer autoantibodies
(approximate geometric mean titer (GMT) of 103)
against TNF-, and coadministration of CFA further boosted titers
10-fold. Ab responses to the foreign (SA) and self (TNF-)
components of the conjugated particle vaccine were similar regardless
of the VLPs used. Both conjugated VLPs induced autoantibody responses
that were 1000-fold higher than when mice were immunized with
unconjugated SA-TNF-3–22 alone (data not
shown and Ref. 12). Thus, structurally similar
polyomavirus and papillomavirus VLPs can function as
autoantibody-inducing conjugated vaccines.
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). These data indicate that conjugation
of rSA to the surface of BPV-L1 VLPs abrogates the specific
interactions with immature DCs that lead to DC activation, and confirm
that DCs are unlikely to be a critical differential mediator of
autoantibody induction by VLPs.
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To determine whether the particulate nature of L1 VLPs is critical
for autoantibody induction, we compared the relative efficiency of
autoantibody induction by VLPs to that of the major subunit of
VLPs, the L1 pentamer. To perform this comparison, we used two HPV16 L1
capsid genes that vary in their ability to form virus-like structures.
Whereas HPV16(K)-L1 self-assembles into full-sized 72-pentamer VLPs,
the assembly-deficient mutant HPV16(P)-L1 has a single amino acid
change in L1 and predominantly forms unassembled pentamers or irregular
aggregates (33). Unlike full-size VLPs, HPV16(P)-L1
pentamers fail to induce neutralizing Abs upon vaccination
(34). HPV16(K)-L1 VLPs or HPV16(P)-L1 pentamers were
generated using a baculovirus expression system and purified and
biotinylated using the same standard methods. Subsequently, HPV16(P)-L1
pentamers were separated from larger irregular aggregates of pentamers
by an additional sucrose gradient centrifugation purification. Although
the extent of biotinylation on L1 pentamers was not directly measured,
we have estimated that conjugated L1 VLPs have 1.5 SA tetramers
bound per L1 molecule, which translates to 540 SA tetramers (and
2160 copies of the TNF- peptide) per VLP (12). By
extension, we predict L1 pentamers to have a minimum of 7.5 SA
tetramers (30 TNF- peptides) per pentamer. This may be an
underestimate, since additional biotin molecules may attach to primary
amines that are accessible on pentamers but not on VLPs because they
are on residues that are normally internal. Even though pentamers are
significantly smaller than VLPs, conjugated pentamers would be
predicted to preserve the same spacing of self-Ag as conjugated
VLPs.
Groups of C57BL/6 mice were immunized with three doses of 5 µg of
HPV16(P)-L1 pentamers or HPV16(K)-L1 VLPs, conjugated to 10 µg of
SA-TNF-3–22, with and without
coadministration of CFA. Preparations were analyzed by Superose 6 gel
filtration chromatography to ensure that the pentamers had not
aggregated in vitro upon conjugation with
SA-TNF-3–22 (11). SA-conjugated
HPV16(P)-L1 pentamers eluted quantitatively in the pentameric fraction
(data not shown). Conjugated pentamers elicited strong autoantibody
responses against TNF-, although they were marginally less effective
(3-fold less) at generating autoantibody responses than conjugated
VLPs (Fig. 3). Responses against the
foreign component of the preparation (SA) were slightly higher (2-fold)
than when conjugated VLPs were used, suggesting that conjugated
pentamers may be somewhat less potent than conjugated VLPs at
diminishing immune distinctions between self and foreign Ags. These
data indicate that the lack of neutralization epitopes on
HPV16(P)-L1 pentamers does not interfere with the ability of conjugated
pentamers to induce autoantibodies. Significantly, the data also
suggest that the particulate nature of the VLP is not critical for
inducing autoantibody responses. However, we cannot definitively rule
out the possibility that pentamers may form aggregates in vivo upon
immunization that may functionally mimic the particulate nature of
wild-type VLPs.
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To examine whether autoantigen spacing influences autoantibody
induction, mice were immunized with VLPs coated with self-Ag at
different densities. To ensure that any differences in immune responses
were not the result of dose effects, the amount of
SA-TNF-3–22 per vaccination was kept
constant, but the protein was dispersed over varying numbers of VLPs.
For example, animals immunized with particles that were completed
coated with SA-TNF-3–22 were injected with 1
µg of biotinylated VLPs conjugated to 2 µg of
SA-TNF-3–22. Density of self-Ag was reduced
to 10%, for example, by conjugating 10 µg of biotinylated VLPs with
2 µg of SA-TNF-3–22 mixed with 18 µg of
rSA. In addition, mice were inoculated with VLPs with self-Ag densities
representing 50 and 20% of maximal occupancy. To ensure that
SA-TNF-3–22 bound biotinylated VLPs
competitively in the presence of rSA, we examined conjugated particles
by ELISA (Fig. 4) and analytical sucrose
gradient (data not shown). The expected amounts of
SA-TNF-3–22 bound to VLPs at all of the
densities tested.
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). Each reduction of self-Ag density had
a corresponding effect of diminishing autoantibody titers. When self-Ag
density was decreased 2-fold (i.e., to 50% maximal occupancy),
autoantibody titers were reduced 5- to 8-fold, depending on whether
adjuvant was used. When mice were immunized with the same dose of
SA-TNF-3–22, but self-Ag was conjugated onto
particles at one-fifth of maximum density, autoantibody titers were
reduced 15- to 40-fold, and when density was reduced to one-tenth of
maximum occupancy, autoantibody titers were reduced 300-fold. At this
density, autoantibody titers were just above background levels and
similar to levels elicited upon immunization with
SA-TNF-3–22 alone (shown here and in Ref.
12). In the absence of CFA, immune responses were lower,
but decreases in self-Ag density had similar relative effects on
autoantibody levels as with CFA.
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15-fold with
the third immunization. At lower occupancies, the effect of boosting
was less pronounced, increasing autoantibody titers 5- to 10-fold at
50 and 25% occupancy, respectively, and 4-fold at 10%
occupancy. Therefore, boosting increased autoantibody titers to a
greater extent in mice immunized with high-occupancy conjugated VLPs
than in those with low-occupancy conjugated VLPs, thereby increasing
the differences in autoantibody titers between these groups.
To address the possibility that competition between SA-specific B cells
and self-Ag-specific B cells might account for the decreased
autoantibody response in mice immunized with particles conjugated at
lower densities, mice were also immunized with a mixture of 1 µg of
L1 VLPs fully conjugated with SA-TNF-3–22 and
9 µg of L1 VLPs fully conjugated to rSA. Immunization with this
mixture elicited high anti-TNF- Ab responses, at levels that
were similar to those in animals immunized with maximally conjugated
SA-TNF- VLPs alone, and 200-fold higher than when mice were
immunized with 10 µg of VLPs at 10% occupancy (Fig. 5
). Thus, in
this situation, immunodominance by the foreign antigenic components of
the vaccine (i.e., L1 or SA) plays a negligible role in diminishing
immune responses against TNF-, indicating that self-Ag density is
the primary determinant in induction of anti-TNF- IgG.
The data described above, when taken together with previous results
demonstrating that vaccination with unconjugated
SA-TNF-3–22 can elicit IgG Ab responses
against SA, suggest further that Ag density may be less important for
inducing Ab responses against foreign Ags than self-Ags at these doses.
To examine how surface density may differentially affect immune
responses to foreign Ags vs self-Ags, we immunized groups of mice with
a constant dose of VLPs (2.5 µg) conjugated with decreasing amounts
of SA-TNF-3–22 in CFA (Fig. 6). When VLPs were coated with saturating
amounts of SA-TNF-3–22, immune responses
against the self and foreign components of the protein were similar
(the ratio of SA to TNF- GMTs was 2). As the amount of
SA-TNF-3–22 on the surface of particles
decreased, Ab responses against both self and foreign declined, but at
different rates and to different degrees. Ab responses against TNF-
declined steadily as the density of Ag decreased. This decrease did not
result from a decrease in the dose of
SA-TNF-3–22 per inoculation, as we observed
little decline in anti-TNF- Ab titers in mice immunized with
lower doses of fully conjugated VLPs. For example, autoantibody titers
declined 2-fold in mice immunized with 0.5 µg vs 2.5 µg of fully
conjugated VLPs (data not shown). In contrast, Ab responses against SA
were relatively stable at the three highest densities tested (and the
ratio of SA to TNF- IgG GMTs increases from 2 to 16 to 64). A
decline in Ab responses against SA was observed only when mice were
immunized with VLPs coated with the lowest amounts of fusion protein.
Conjugation of SA-TNF-3–22 to VLPs can
increase Ab responses to SA (there is a 5-fold boost in Ab titers after
conjugation at high density, as seen in Fig. 5), so this decline in Ab
responses to SA at low dose/low occupancy may, in part, reflect a loss
of this enhancing effect. Therefore, although high Ag density is
critical for generating high-titer IgG Ab responses against self, it
provides only a moderate quantitative boost in IgG Ab responses against
foreign.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We demonstrated that self-peptides conjugated to papillomavirus and polyomavirus VLPs induced similar IgG autoantibody responses. The fact that polyomavirus VLPs do not induce acute activation of BMDCs (24), coupled with our data demonstrating that SA conjugation abrogates the ability of papillomavirus VLPs to activate BMDCs, indicates that direct activation of DCs by the immunogen does not play a critical role in autoreactive B cell responses. Presumably, any highly ordered Ag with repetitive, dense spacing (including other viruses, bacterial pili, or synthetic particles) could function as conjugated immunogens to induce autoantibody responses. Consistent with this idea, Fehr et al. (18) reported that Ab bound at high density to vesicular stomatitis virus particles or UV-inactivated bacteria (Salmonella typhi and Pseudomonas aeruginosa) induced anti-Ig autoantibody responses.
To further define the requirements for autoimmune activation, we
performed experiments examining the effects of Ag size and density on
autoantibody induction. Somewhat surprisingly, conjugated HPV16(P)-L1
pentamers induced autoantibodies at only a slightly reduced efficiency
relative to full-size 72-pentamer VLPs. Although pentamers are
predicted to have the same spacing of self-Ag, they may contain as few
as 30 TNF- peptides. This analysis indicates that a large
particulate Ag is not required for effective autoantibody induction,
although it does not establish a lower limit for the number of repeated
epitopes needed to accomplish this.
High Ag density was critical for autoantibody induction. In experiments
in which we controlled for dose and the possibility of immune dominance
by competing foreign antigenic epitopes, we observed that reducing the
density of self-Ag on VLP surfaces dramatically diminished autoantibody
responses. Based on our previous estimate of 1.5 SA-TNF- tetramers
per L1 molecule, we calculate that the tetramers on particles which are
fully conjugated are spaced 60 Å apart (data not shown). This
distance increases to 85 Å at 50% density, 140 Å at 20% density,
and 195 Å at 10% density. Our finding that autoantibody responses are
strongest to conjugated VLPs with 100 and 50% occupancy, but decrease
rapidly in response to immunization with lower occupancy particles,
supports the previous proposal that the immune system has evolved to
respond strongly to Ags with an epitope spacing of 50–100 Å
(13, 14, 15). This spacing is often found on microbial
surfaces, but seldom in their vertebrate hosts. B cells may have
evolved to recognize repetitive Ags with this particular spacing as
foreign or dangerous. Our results indicate that B cell recognition of
"foreign" structural elements is immunodominant over mechanisms
that normally maintain tolerance to soluble central self-Ags. It would
be interesting to determine whether further increases in Ag density
(that are technically unfeasible using our conjugation system) would
result in a further increase, or conversely a decrease, in autoantibody
responses. In contrast to autoantibody induction, strong Ab responses
against SA can be induced by immunization with
SA-TNF-3–22 conjugated to VLPs at lower
densities, as well as with unconjugated
SA-TNF-3–22 tetramers. These data suggest
that the signaling required to produce B cell responses specific for
foreign Ags at these doses is distinct from, or less stringent than,
that required to overcome B cell tolerance.
Numerous studies have previously shown that the repetitiveness of
foreign Ags, ranging from bacterial flagellin (35),
viruses and viral capsids (36, 37, 38), to haptenated polymers
(39), can strongly influence B cell responses to foreign
epitopes. Binding of repetitively arranged Ags to B cell receptors
obviates the need for second signals to induce B cell responses. Thus,
induction of IgM, but not class switching to IgG, is independent of T
cell help for this class of Ags. T-independent B cell activation has
also been correlated with Ag density. For instance, high-density
haptenated beads acted as T-independent Ags and low-density haptenated
beads acted as T-dependent Ags (35). Thus, it appears that
T-independent and autoantibody responses are promoted by similar
antigenic structures. However, this correlation is apparently not due
to a limitation of T help in our experimental system for breaking B
cell tolerance. The SA polypeptide clearly contains Th epitopes that
would provide linked T help for the TNF- autoantigen. This is
exemplified by the strong IgG response to the SA portion of the fusion
protein, even without conjugation to the VLPs. The similar structural
requirements for the induction of autoreactive and T-independent B cell
responses may be due to a similar stringent requirements for Ag-induced
cross-linking of B cell receptors. Whether the critical downstream
signals are the same or distinct for these two types of responses
remains to be determined.
Interactions between potentially autoreactive B cells and repetitive self-Ags could potentially break tolerance at several stages during the development of the B cell responses, from the initial activation of autoreactive B cells to their proliferation in lymphoid tissues, or for autoreactive B cell survival or escape from anergy after encountering self-Ag in its normal context. Because previous data from our laboratory indicated that both irregularly and repetitively displayed self-Ags elicit similar levels of self-reactive IgM early after immunization (12), we do not favor the idea that the initial B cell activation is the critical point at which Ag structure influences autoantibody responses. Because only densely arrayed self-Ag induced efficient IgG production, we instead favor the hypothesis that self-reactive mature B cells preferentially escape anergy that normally results upon encountering self-Ag in its normal context (5) if they have previously been activated by densely ordered self-Ag. It is also possible that self-Ag-induced anergic B cells could be reactivated upon subsequent interactions with repetitive self-Ag (40).
Mechanistic studies of how repetitive Ags promote autoantibody responses could provide insights into how autoreactive B cells arise in autoimmune diseases. In addition, these studies could further the development of autoantibody-inducing vaccines against the mediators of several important diseases, including arthritis, allergies, cancer, and Alzheimer disease.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: VLP, virus-like particle; SA, streptavidin; DC, dendritic cell; BPV, bovine papillomavirus; HPV, human papillomavirus; BKV, BK virus; BMDC, bone marrow-derived DC; GMT, geometric mean titer.
Received for publication July 10, 2002. Accepted for publication September 19, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Nat. Biotechnol. 17:666.[Medline]
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R. Yang, F. M. Murillo, M. J. Delannoy, R. L. Blosser, W. H. Yutzy IV, S. Uematsu, K. Takeda, S. Akira, R. P. Viscidi, and R. B. S. Roden B Lymphocyte Activation by Human Papillomavirus-Like Particles Directly Induces Ig Class Switch Recombination via TLR4-MyD88 J. Immunol., June 15, 2005; 174(12): 7912 - 7919. [Abstract] [Full Text] [PDF]
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R. Yang, F. M. Murillo, H. Cui, R. Blosser, S. Uematsu, K. Takeda, S. Akira, R. P. Viscidi, and R. B. S. Roden Papillomavirus-Like Particles Stimulate Murine Bone Marrow-Derived Dendritic Cells To Produce Alpha Interferon and Th1 Immune Responses via MyD88 J. Virol., October 15, 2004; 78(20): 11152 - 11160. [Abstract] [Full Text] [PDF]
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R. Yang, F. M. Murillo, K.-Y. Lin, W. H. Yutzy IV, S. Uematsu, K. Takeda, S. Akira, R. P. Viscidi, and R. B. S. Roden Human Papillomavirus Type-16 Virus-Like Particles Activate Complementary Defense Responses in Key Dendritic Cell Subpopulations J. Immunol., August 15, 2004; 173(4): 2624 - 2631. [Abstract] [Full Text] [PDF]
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B. Chackerian, L. Briglio, P. S. Albert, D. R. Lowy, and J. T. Schiller Induction of Autoantibodies to CCR5 in Macaques and Subsequent Effects upon Challenge with an R5-Tropic Simian/Human Immunodeficiency Virus J. Virol., April 15, 2004; 78(8): 4037 - 4047. [Abstract] [Full Text] [PDF]
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 C. A. Velikovsky, F. A. Goldbaum, J. Cassataro, S. Estein, R. A. Bowden, L. Bruno, C. A. Fossati, and G. H. Giambartolomei Brucella Lumazine Synthase Elicits a Mixed Th1-Th2 Immune Response and Reduces Infection in Mice Challenged with Brucella abortus 544 Independently of the Adjuvant Formulation Used Infect. Immun., October 1, 2003; 71(10): 5750 - 5755. [Abstract] [Full Text] [PDF]
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 H. Schellekens Immunogenicity of therapeutic proteins Nephrol. Dial. Transplant., July 1, 2003; 18(7): 1257 - 1259. [Full Text] [PDF]
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) and rSA (
) were determined by ELISA.
Mice were immunized with or without coadministration of CFA, as
indicated. Data represent the GMTs from groups of three mice. Error
bars represent SEM.
